His first prime time role came in 1994, in the soap opera Pátria Minha. Poor ratings and reviews caused the production to be shortened, but Santoro managed to make enough money to be able to rent his own flat again, this time in the upscale neighbourhood of Leblon, where he could often be seen jogging by the beach.

In 1996, he was cast as the male lead character in the soap opera O Amor Está no Ar. The production, which involved an intergeneration love triangle and extraterrestrial beings, brought him some trouble: his performances and the soap itself weren't greatly praised by critics, but the hectic filming schedule forced him to quit college. Santoro made his big screen debut that same year, in the acclaimed short Depois do Escuro.

He gained further big screen experience with a part in the 1998 movie Como Ser Solteiro, which was somewhat obscured by his critically acclaimed performance as Frei Malthus in the TV Globo mini-series Hilda Furacão. Until then, he had been regarded as merely a capable actor, and his talent for more serious and prominent roles had often been questioned by critics. Santoro has stated that working on Hilda Furacão was one of the toughest experiences in his career, due to the dramatic load of the character, a young Franciscan priest who falls in love with Hilda, a prostitute.

Santoro was the voice of Stuart Little in the Brazilian release of the 1999 film, as well as its 2002 sequel. Also in 1999, he played a prominent part in the ill-fated soap Suave Veneno, and a small part in the comedy film O Trapalhão e a Luz Azul, by Brazilian comedian Renato Aragão.

His first major role in a cinema production would come in 2001, with Bicho de Sete Cabeças (Brainstorm) by Laís Bodansky. At the time, he was under heavy criticism for his role in the soap Estrela-Guia and was even booed at the first showing of the film at the 2000 Brasília Brazilian Cinema Festival. By the end of the film, he received a standing ovation from the audience and critics praised his portrayal of a young man who is sent to a psychiatric clinic by his father after being caught smoking marijuana. He also went on to win the festival's best actor trophy.

After Bicho, his reputation as an actor had been solidified and he was cast as the male lead in the much-praised Abril Despedaçado (Behind the Sun), one of the nominees for best foreign film in the 2002 Golden Globe Awards. He had another critically acclaimed performance in the 2003 Hector Babenco film Carandiru, where he played a transexual prisoner known as Lady Di.

Canadian director Robert Allan Ackerman signed Santoro for his TV production The Roman Spring of Mrs. Stone after being mesmerised by his performance on Bicho de Sete Cabeças. Since Santoro didn't have an agent in North America, Ackerman contacted Santoro's father. He spent two months filming in Rome, alongside renowned actors such as Helen Mirren and Anne Bancroft. Soon after he finished shooting for "Mrs. Stone", he received an offer from Columbia Pictures for a part in the blockbuster Charlie's Angels: Full Throttle.

His performance in "Charlie's Angels" kick-started his career in Hollywood. After "Charlie's Angels," he played the character of Karl, the "enigmatic chief designer" and love interest of Laura Linney's character, in the romantic comedy Love Actually, alongside Hugh Grant, Emma Thompson, Keira Knightley and Rowan Atkinson. He also started getting work in the advertising business as he appeared in a Credicard's campaign called "the Best of the Life" with Gisele Bundchen in 2002 and in a 2-minute-long commercial for Chanel, directed by Baz Luhrmann alongside Nicole Kidman in 2004. In 2006, Santoro joined the cast of Lost as survivor of Oceanic Flight 815, Paulo. His first appearance was in episode titled, "Further Instructions". Despite his successful international career, Santoro has said he doesn't want to move to Los Angeles and would like to stay in Rio. He practices transcendental meditation and sports when time allows, is a fan of The Doors and likes to read William Shakespeare's works. His favorite actors are Robert De Niro and Al Pacino.

Santoro's name was added to the main cast in A Tale of Two Cities, the first episode of Season 3. Exposé was the last episode in which he was credited as a part of the main cast, and he hasn't been seen on the show since.